Serpin fusion polypeptides and methods of use thereof

ABSTRACT

This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin and second polypeptide comprising of at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP containing polypeptide; and an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide. This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/536,976, filed Jun. 28, 2012, which claims the benefit of U.S.Provisional Application No. 61/502,055, filed Jun. 28, 2011; U.S.Provisional Application No. 61/570,394, filed Dec. 14, 2011; and U.S.Provisional Application No. 61/577,204, filed Dec. 19, 2011; and U.S.Provisional Application No. 61/638,168, filed Apr. 25, 2012. Thecontents of each of these applications are hereby incorporated byreference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “INHI002C02US_SeqList.txt”, whichwas created on Feb. 6, 2015 and is 149 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to molecules, particularly polypeptides, moreparticularly fusion proteins that include a serpin polypeptide or anamino acid sequence that is derived from a serpin polypeptides and asecond polypeptide. Additionally, the invention relates to fusionproteins that include a serpin polypeptide or an amino acid sequencethat is derived from serpin polypeptides, a second polypeptide, and athird polypeptide. Specifically, this invention relates to fusionproteins that include at least one serpin polypeptide and a secondpolypeptide or fusion proteins that include at least one serpinpolypeptide, a second polypeptide, and a third polypeptide, where thesecond and third polypeptides of the fusion proteins of the inventioncan be at least one the following: an Fc polypeptide or an amino acidsequence that is derived from an Fc polypeptide; a cytokine targetingpolypeptide or a sequence derived from a cytokine targeting polypeptide;a WAP domain containing polypeptide or a sequence derived from a WAPcontaining polypeptide; or an albumin polypeptide or an amino acidsequence that is derived from a serum albumin polypeptide. Thisinvention also relates to methods of using such molecules in a varietyof therapeutic and diagnostic indications, as well as methods ofproducing such molecules.

BACKGROUND OF THE INVENTION

Aberrant serine protease activity or an imbalance ofprotease-to-protease inhibitor can lead to protease-mediated tissuedestruction and inflammatory responses. Accordingly, there exists a needfor therapeutics and therapies that target aberrant serine proteaseactivity and/or imbalance of protease-to-protease inhibitor.Furthermore, enhanced therapeutic effects may be gained through theattenuation of aberrant cytokine signaling and serine protease activity.In addition, serpin proteins have demonstrated anti-infective activitieswhile targeting inflammatory cytokines has been shown to increase therisk of infection. The fusion proteins of this invention have thepotential to dampen inflammatory cytokine activity and limit the risk ofinfection.

SUMMARY OF THE INVENTION

The fusion proteins described herein include at least a serpinpolypeptide or an amino acid sequence that is derived from a serpinpolypeptide (Polypeptide 1) and second polypeptide (Polypeptide 2).Additionally, the fusion proteins described herein include a serpinpolypeptide or an amino acid sequence that is derived from a serpinpolypeptide (Polypeptide 1), a second polypeptide (Polypeptide 2), and athird polypeptide (Polypeptide 3). As used interchangeably herein, theterms “fusion protein” and “fusion polypeptide” refer to a serpinpolypeptide or an amino acid sequence derived from a serpin polypeptideoperably linked to at least a second polypeptide or an amino acidsequence derived from at least a second polypeptide. The individualizedelements of the fusion protein can be linked in any of a variety ofways, including for example, direct attachment, the use of anintermediate or a spacer peptide, the use of a linker region, the use ofa hinge region or the use of both a linker and a hinge region. In someembodiments, the linker region may fall within the sequence of the hingeregion, or alternatively, the hinge region may fall within the sequenceof the linker region. Preferably, the linker region is a peptidesequence. For example, the linker peptide includes anywhere from zero to40 amino acids, e.g., from zero to 35 amino acids, from zero to 30 aminoacids, from zero to 25 amino acids, or from zero to 20 amino acids.Preferably, the hinge region is a peptide sequence. For example, thehinge peptide includes anywhere from zero to 75 amino acids, e.g., fromzero to 70 amino acids, from zero to 65 amino acids or from zero to 62amino acids. In embodiments where the fusion protein includes both alinker region and hinge region, preferably each of the linker region andthe hinge region is a peptide sequence. In these embodiments, the hingepeptide and the linker peptide together include anywhere from zero to 90amino acids, e.g., from zero to 85 amino acids or from zero to 82 aminoacids.

In some embodiments, the serpin polypeptide and the second polypeptidecan be linked through an intermediate binding protein. In someembodiments, the serpin-based portion and second polypeptide portion ofthe fusion protein may be non-covalently linked.

In some embodiments, fusion proteins according to the invention can haveone of the following formulae, in an N-terminus to C-terminus directionor in a C-terminus to N-terminus direction:

-   -   Polypeptide 1_((a))-hinge_(m)-Polypeptide 2_((b))    -   Polypeptide 1_((a))-linker_(n)-Polypeptide 2_((b))    -   Polypeptide 1_((a))-linker_(n)-hinge_(m)-Polypeptide 2_((b))    -   Polypeptide 1_((a))-hinge_(m)-linker_(n)-Polypeptide 2_((b))    -   Polypeptide 1_((a))-Polypeptide 2_((b))-Polypeptide 3_((c))    -   Polypeptide 1_((a))-hinge_(m)-Polypeptide        2_((b))-hinge_(m)-Polypeptide 3_((c))    -   Polypeptide 1_((a))-linker_(n)-Polypeptide        2_((b))-linker_(n)-Polypeptide 3_((c))    -   Polypeptide 1_((a))-hinge_(m)-linker_(n)-Polypeptide        2_((b))-hinge_(m)-linker_(n)-Polypeptide 3_((c))    -   Polypeptide 1_((a))-linker_(n)-hinge_(m)-Polypeptide        2_((b))-linker_(n)-hinge_(m)-Polypeptide 3_((c))    -   where n is an integer from zero to 20, where m is an integer        from 1 to 62 and where a, b, and c integers of at least 1. These        embodiments include the above formulations and any variation or        combination thereof. For example, the order of polypeptides in        the formulae also includes Polypeptide 3_((c))-Polypeptide        1_((a))-Polypeptide 2_((b)), Polypeptide 2_((b))-Polypeptide        3_((c))-Polypeptide 1_((a)), or any variation or combination        thereof.

In some embodiments, the Polypeptide 1 sequence includes a serpinpolypeptide. Serpins are a group of proteins with similar structuresthat were first identified as a set of proteins able to inhibitproteases. Serpin proteins suitable for use in the fusion proteinsprovided herein include, by way of non-limiting example, alpha-1antitrypsin (AAT), antitrypsin-related protein (SERPINA2), alpha1-antichymotrypsin (SERPINA3), kallistatin (SERPINA4), monocyteneutrophil elastase inhibitor (SERPINB1), PI-6 (SERPINB6), antithrombin(SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha2-antiplasmin (SERPINF2), complement 1-inhibitor (SERPING1), andneuroserpin (SERPINI1).

In some embodiments, the Polypeptide 1 sequence includes an alpha-1antitrypsin (AAT) polypeptide sequence or an amino acid sequence that isderived from AAT. In some embodiments, the Polypeptide 1 sequenceincludes a portion of the AAT protein. In some embodiments, thePolypeptide 1 sequence includes at least the reactive site loop portionof the AAT protein. In some embodiments, the reactive site loop portionof the AAT protein includes at least the amino acid sequence:GTEAAGAMFLEAIPMSIPPEVKFNK SEQ ID NO: 1).

In a preferred embodiment, the AAT polypeptide sequence or an amino acidsequence that is derived from AAT is or is derived from a human AATpolypeptide sequence.

In some embodiments, the fusion protein includes a full-length human AATpolypeptide sequence having the following amino acid sequence:

(SEQ ID NO: 2) 1EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA 61FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL 121SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT 181VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL 241LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI 361PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK

In some embodiments, the fusion protein includes a human AAT polypeptidesequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 2.

In some embodiments, the AAT polypeptide sequence is, or the amino acidsequence derived from an AAT polypeptide is derived from, one or more ofthe human AAT polypeptide sequences shown in GenBank Accession Nos.AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1,NP_(—)001002235.1, CAA34982.1, NP_(—)001002236.1, NP_(—)000286.3,NP_(—)001121179.1, NP_(—)001121178.1, NP_(—)001121177.1,NP_(—)001121176.16, NP_(—)001121175.1, NP_(—)001121174.1,NP_(—)001121172.1, and/or AAA51547.1.

In some embodiments, the fusion proteins contain one or more mutations.For example, the fusion protein contains at least one mutation at amethionine (Met) residue in the serpin portion of the fusion protein. Inthese Met mutations, the Met residue can be substituted with any aminoacid. For example, the Met residue can be substituted with an amino acidwith a hydrophobic side chain, such as, for example, leucine (Leu, L).Without wishing to be bound by theory, the Met mutation(s) preventoxidation and subsequent inactivation of the inhibitory activity of thefusion proteins of the invention. In some embodiments, the Met residuecan be substituted with a charged residue, such as, for example,glutamate (Glu, E). In some embodiments, the Met mutation is at position358 of an AAT polypeptide. For example, the Met mutation is Met358Leu(M358L). In some embodiments, the Met mutation is at position 351 of anAAT polypeptide. For example, the Met mutation is Met351Glu (M351E). Insome embodiments, the Met mutation is at position 351 and at position358 of an AAT polypeptide, for example, the Met mutation is Met351Glu(M351E) and Met358Leu (M358L). For example, the reactive site loop ofthis variant of the fusion AAT polypeptide has the following sequence:GTEAAGAEFLEAIPLSIPPEVKFNK (SEQ ID NO: 32). In some embodiments, the Metmutation is at position 351 and at position 358 of an AAT polypeptide,for example, the Met mutation is Met351Leu (M351L) and Met358Leu(M358L). For example, the reactive site loop of this variant of thefusion AAT polypeptide has the following sequence:GTEAAGALFLEAIPLSIPPEVKFNK (SEQ ID NO: 33).

In some embodiments, the second polypeptide (Polypeptide 2) of theserpin fusion protein is an Fc polypeptide or derived from an Fcpolypeptide. These embodiments are referred to collectively herein as“serpin-Fc fusion proteins.” The serpin-Fc fusion proteins describedherein include at least a serpin polypeptide or an amino acid sequencethat is derived from a serpin and an Fc polypeptide or an amino acidsequence that is derived from an Fc polypeptide. In some embodiments,the serpin-Fc fusion protein includes a single serpin polypeptide. Inother embodiments, the serpin-Fc fusion proteins includes more than oneserpin polypeptide, and these embodiments are collectively referred toherein as “serpin_((a′))-Fc fusion protein,” wherein (a′) is an integerof at least 2. In some embodiments, each serpin polypeptides in aserpin_((a′))-Fc fusion protein includes the same amino acid sequence.In other embodiments, each serpin polypeptide in a serpin_((a′))-Fcfusion protein includes serpin polypeptides with distinct amino acidsequences. The serpin polypeptides of serpin_((a′))-Fc fusion proteinscan be located at any position within the fusion protein.

In some embodiments, the serpin polypeptide of the serpin-Fc fusionprotein includes at least the amino acid sequence of the reactive siteloop portion of the AAT protein. In some embodiments, the reactive siteloop portion of the AAT protein includes at least the amino acidsequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide ofthe serpin-Fc fusion protein includes at least the amino acid sequenceof a variant of the reactive site loop portion of the AAT protein. Insome embodiments, the variant of the reactive site loop portion of theAAT protein includes at least the amino acid sequence of SEQ ID NO:32 orSEQ ID NO:33. In some embodiments, the serpin polypeptide of theserpin-Fc fusion protein includes at least the full-length human AATpolypeptide sequence having amino acid sequence of SEQ ID NO: 2. In someembodiments the serpin polypeptide of the serpin-Fc fusion proteinincludes human AAT polypeptide sequence that is at least 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the serpin-Fc fusionprotein includes the AAT polypeptide sequence is or the amino acidsequence derived from an AAT polypeptide is derived from one or more ofthe human AAT polypeptide sequences shown in GenBank Accession Nos.AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1,NP_(—)001002235.1, CAA34982.1, NP_(—)001002236.1, NP_(—)000286.3,NP_(—)001121179.1, NP_(—)001121178.1, NP_(—)001121177.1,NP_(—)001121176.16, NP_(—)001121175.1, NP_(—)001121174.1,NP_(—)001121172.1, and/or AAA51547.1.

In some embodiments, the Fc polypeptide of the fusion protein is a humanFc polypeptide, for example, a human IgG Fc polypeptide sequence or anamino acid sequence that is derived from a human IgG Fc polypeptidesequence. For example, in some embodiments, the Fc polypeptide is ahuman IgG1 Fc polypeptide or an amino acid sequence that is derived froma human IgG1 Fc polypeptide sequence. In some embodiments, the Fcpolypeptide is a human IgG2 Fc polypeptide or an amino acid sequencethat is derived from a human IgG2 Fc polypeptide sequence. In someembodiments, the Fc polypeptide is a human IgG3 Fc polypeptide or anamino acid sequence that is derived from a human IgG3 Fc polypeptidesequence. In some embodiments, the Fc polypeptide is a human IgG4 Fcpolypeptide or an amino acid sequence that is derived from a human IgG4Fc polypeptide sequence. In some embodiments, the Fc polypeptide is ahuman IgM Fc polypeptide or an amino acid sequence that is derived froma human IgM Fc polypeptide sequence.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG1 Fc polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 3) 1APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 61PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 3.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide is mutated or modified to enhanceFcRn binding. In these embodiments the mutated or modified Fcpolypeptide includes the following mutations: Met252Tyr, Ser254Thr,Thr256Glu (M252Y, S256T, T256E) or Met428Leu and Asn434Ser (M428L,N434S) using the Kabat numbering system. In some embodiments the Fcpolypeptide portion is mutated or otherwise modified so as to disruptFc-mediated dimerization. In these embodiments, the fusion protein ismonomeric in nature.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG2 Fc polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 4) 1APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP 61REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL 121PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT 181VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 4.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG3 Fc polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 5) 1APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK 61PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT 121LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL 181TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 5.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG4 Fc polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 6) 1APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK 61PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121LPPSQEEMTK NQVSLTCLVK GFYPDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT 181VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 6.

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgM Fc polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 7) 1IAELPPKVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ VSWLREGKQV GSGVTTDQVQ 61AEAKESGPTT YKVTSTLTIK ESDWLGQSMF TCRVDHRGLT FQQNASSMCV PDQDTAIRVF 121AIPPSFASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE AVKTHTNISE SHPNATFSAV 181GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG

In some embodiments where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide of the fusion protein includes ahuman IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments of the fusion proteins provided herein, the secondpolypeptide (Polypeptide 2) of the serpin fusion protein is a cytokinetargeting polypeptide or derived from a cytokine targeting polypeptide.These embodiments are referred to collectively herein as“serpin-cytokine targeting polypeptide fusion proteins.” Theserpin-cytokine targeting polypeptide fusion proteins described hereininclude at least a serpin polypeptide or an amino acid sequence that isderived from a serpin polypeptide and a cytokine targeting polypeptide,or derivation thereof. In some embodiments, the serpin-cytokinetargeting polypeptide fusion protein includes a single serpinpolypeptide. In other embodiments, the serpin-cytokine targetingpolypeptide fusion protein includes more than one serpin polypeptide,and these embodiments are collectively referred to herein as“serpin_((a′))-cytokine targeting polypeptide fusion proteins,” wherein(a′) is an integer of at least 2. In some embodiments, each serpinpolypeptide in a serpin_((a′))-cytokine targeting polypeptide fusionprotein includes the same amino acid sequence. In other embodiments,each serpin polypeptide of a serpin_((a))-cytokine targeting polypeptidefusion protein includes serpin polypeptides with distinct amino acidsequences.

In some embodiments, the cytokine targeting polypeptide of theserpin-cytokine targeting polypeptide fusion protein is a cytokinereceptor or derived from a cytokine receptor. In a preferred embodiment,the cytokine targeting polypeptide or an amino acid sequence that isderived from the cytokine receptor is or is derived from a humancytokine receptor sequence. In other embodiments, the cytokine targetingpolypeptide is an antibody or an antibody fragment, for example ananti-cytokine antibody or anti-cytokine antibody fragment. In apreferred embodiment, the cytokine targeting polypeptide or an aminoacid sequence that is derived from the antibody or antibody fragment isderived from a chimeric, humanized, or fully human antibody sequence.The term antibody fragment includes single chain, Fab fragment, aF(ab′)₂ fragment, a scFv, a scAb, a dAb, a single domain heavy chainantibody, and a single domain light chain antibody.

In other embodiments, the cytokine targeting polypeptide binds acytokine receptor and prevents binding of a cytokine to the receptor. Inother embodiments, the cytokine targeting polypeptide is an antibody oran antibody fragment, for example an anti-cytokine receptor antibody oranti-cytokine receptor antibody fragment.

In some embodiments, the serpin polypeptide of the serpin-cytokinetargeting polypeptide fusion proteins includes at least the amino acidsequence of the reactive site loop portion of the AAT protein. In someembodiments, the reactive site loop portion of the AAT protein includesat least the amino acid sequence of SEQ ID NO:1. In some embodiments,the serpin polypeptide of the serpin-cytokine targeting fusion proteinsincludes at least the amino acid sequence of a variant of the reactivesite loop portion of the AAT protein. In some embodiments, the variantof the reactive site loop portion of the AAT protein includes at leastthe amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In someembodiments, the serpin polypeptide of the serpin-cytokine targetingfusion protein includes or is derived from at least the full-lengthhuman AAT polypeptide sequence having amino acid sequence of SEQ ID NO:2. In some embodiments the serpin polypeptide of the serpin-cytokinetargeting fusion protein includes human AAT polypeptide sequence that isat least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the serpin-cytokinetargeting fusion protein includes an AAT polypeptide sequence or anamino acid sequence derived from an AAT polypeptide that is or isderived from one or more of the human AAT polypeptide sequences shown inGenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1,AAA51546.1, CAA25838.1, NP_(—)001002235.1, CAA34982.1,NP_(—)001002236.1, NP_(—)000286.3, NP_(—)001121179.1, NP_(—)001121178.1,NP_(—)001121177.1, NP_(—)001121176.16, NP_(—)001121175.1,NP_(—)001121174.1, NP_(—)001121172.1, and/or AAA51547.1.

The serpin-cytokine targeting polypeptide fusion protein can incorporatea portion of the serpin-Fc fusion protein. For example, an antibodycontains an Fc polypeptide. Therefore, in some embodiments where thecytokine targeting polypeptide is a cytokine-targeting antibody, theserpin-cytokine targeting polypeptide fusion protein will incorporate aportion of the serpin-Fc fusion protein. Furthermore, most receptorfusion proteins that are of therapeutic utility are Fc fusion proteins.Thus, in some embodiments, wherein the serpin-cytokine targetingpolypeptide fusion protein is a serpin-cytokine receptor fusion protein,the serpin-cytokine targeting polypeptide fusion protein may incorporatean Fc polypeptide in addition to the serpin portion and the cytokinereceptor portion.

In some embodiments, where the serpin-cytokine targeting polypeptidefusion protein includes an Fc polypeptide sequence, the Fc polypeptidesequence includes or is derived from the amino acid sequence of any oneof SEQ ID NO: 3, 4, 5, 6, or 7. In some embodiments where theserpin-cytokine targeting fusion protein includes an Fc polypeptidesequence, the Fc polypeptide sequence has at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to any one of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6,or 7. In some embodiments, the serpin polypeptide and the cytokinetargeting polypeptide are operably linked via a linker region, forexample, a glycine-serine linker or glycine-serine based linker. In someembodiments, the serpin polypeptide and the cytokine targetingpolypeptide are operably linked via a hinge region. In some embodiments,the serpin polypeptide and the cytokine targeting polypeptide areoperably linked via a linker region and a hinge region. In otherembodiments, the serpin polypeptide and the cytokine targetingpolypeptide are directly attached.

In some embodiments of the fusion proteins provided herein, the secondpolypeptide (Polypeptide 2) of the serpin fusion protein is a wheyacidic protein (WAP) domain containing polypeptide, or an amino acidsequence that is derived from a WAP domain containing polypeptide. Theseembodiments are referred to collectively herein as “serpin-WAP domainfusion proteins.” The serpin-WAP domain fusion proteins include at leasta serpin polypeptide or at least an amino acid sequence that is derivedfrom a serpin, a WAP domain-containing polypeptide or an amino acidsequence that is derived from a WAP domain-containing polypeptide. Insome embodiments, the serpin-WAP domain fusion protein includes a singleserpin polypeptide. In other embodiments, the serpin-WAP targetingpolypeptide fusion protein includes more than one serpin polypeptide.These embodiments are collectively referred to herein as“serpin_((a′))-WAP domain fusion proteins,” wherein (a′) is an integerof at least 2. In some embodiments, serpin polypeptides of theserpin_((a′))-WAP domain fusion protein includes the same amino acidsequence. In other embodiments, the serpin polypeptides of theserpin_((a′))-cytokine targeting polypeptide fusion protein, includesserpin polypeptides with distinct amino acid sequences.

These serpin-WAP domain fusion proteins include a WAP domain containingpolypeptide or polypeptide sequence that is or is derived from a WAPdomain containing polypeptide. The WAP domain is an evolutionarilyconserved sequence motif of 50 amino acids containing eight cysteinesfound in a characteristic 4-disulfide core arrangement (also called afour-disulfide core motif). The WAP domain sequence motif is afunctional motif characterized by serine protease inhibition activity ina number of proteins.

WAP domain-containing polypeptides suitable for use in the fusionproteins provided herein include, by way of non-limiting example,secretory leukocyte protease inhibitor (SLPI), Elafin, and Eppin.

In some embodiments, the WAP domain-containing polypeptide sequence ofthe fusion protein includes a secretory leukocyte protease inhibitor(SLPI) polypeptide sequence or an amino acid sequence that is derivedfrom SLPI. These embodiments are referred to herein as“serpin-SLPI-derived fusion proteins.” In some embodiments, the SLPIpolypeptide sequence comprises a portion of the SLPI protein, such asfor example, the WAP2 domain or a sub-portion thereof. In a preferredembodiment, the SLPI polypeptide sequence or an amino acid sequence thatis derived from SLPI is or is derived from a human SLPI polypeptidesequence.

In some embodiments of the serpin-SLPI fusion proteins of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes a full-length human SLPI polypeptide sequence having thefollowing amino acid sequence:

(SEQ ID NO: 8) 1MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK 61KRCCPDTCGI KCLDPVDTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG 121MCGKSCVSPV KA

In some embodiments of the serpin-SLPI fusion protein of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes a human SLPI polypeptide sequence that is at least 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 8.

In some embodiments of the serpin-SLPI fusion protein of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes a portion of the full-length human SLPI polypeptide sequence,where the portion has the following amino acid sequence:

(SEQ ID NO: 9) 1SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK 61PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA

In some embodiments of the serpin-SLPI fusion protein of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes a human SLPI polypeptide sequence that is at least 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 9.

In some embodiments of the serpin-SLPI fusion protein of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes the WAP2 domain of the full-length human SLPI polypeptidesequence, where the WAP2 domain has the following amino acid sequence:

(SEQ ID NO: 10) 1TRRKPGKCPV TYGQCLMLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A

In some embodiments of the serpin-SLPI fusion protein of the invention,the SLPI sequence or a SLPI-derived sequence of the fusion proteinincludes a human SLPI polypeptide sequence that is at least 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 10.

In some embodiments of the serpin-SLPI fusion proteins of the invention,the SLPI polypeptide sequence or the amino acid sequence derived from anSLPI polypeptide is or is derived from, one or more of the human SLPIpolypeptide sequences shown in GenBank Accession Nos. CAA28187.1,NP_(—)003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1,CAA28188.1, AAD19661.1, and/or BAG35125.1.

In some embodiments of the serpin-SLPI fusion proteins of the invention,the SLPI polypeptide sequence or a SLPI-derived sequence of the fusionprotein includes a human SLPI polypeptide sequence that is modified at aMethoine (Met) residue. In these Met mutations, the Met residue can besubstituted with any amino acid. For example, the Met residue can besubstituted with an amino acid with a hydrophobic side chain, such as,for example, leucine (Leu, L) or valine (Val, V). Without wishing to bebound by theory, the Met mutation(s) prevent oxidation and subsequentinactivation of the inhibitory activity of the fusion proteins of theinvention. In some embodiments, the Met mutation is at position 98 of anSLPI polypeptide. For example, the modified SLPI polypeptide sequence ofthe serpin-SLPI includes mutations M98L or M98V in SEQ ID NO: 8.

In other embodiments, the WAP domain-containing polypeptide sequence ofthe fusion protein includes an elafin polypeptide sequence or an aminoacid sequence that is derived from elafin. These embodiments arereferred to herein as “serpin-elafin fusion proteins. In someembodiments, the elafin polypeptide sequence includes a portion of theelafin protein, such as for example, the WAP domain or a sub-portionthereof. In a preferred embodiment, the elafin polypeptide sequence oran amino acid sequence that is derived from elafin is or is derived froma human elafin polypeptide sequence.

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes a full-length human elafin polypeptide sequence havingthe following amino acid sequence:

(SEQ ID NO: 11) 1MRASSFLIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK 61AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes a human elafin polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11.

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes a portion of the full-length human elafin polypeptidesequence, where the portion has the following amino acid sequence:

(SEQ ID NO: 12) 1AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR 61CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes a human elafin polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes the WAP domain of the full-length human elafinpolypeptide sequence, where the WAP domain has the following amino acidsequence:

(SEQ ID NO: 13) 1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ

In some embodiments of the serpin-elafin fusion proteins, the fusionprotein includes a human elafin polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13.

In some embodiments of the serpin-elafin fusion proteins, the elafinpolypeptide sequence or the amino acid sequence derived from an elafinpolypeptide is derived from one or more of the human elafin polypeptidesequences shown in GenBank Accession Nos. P19957.3, NP_(—)002629.1,BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and/or NP_(—)542181.1.

In other embodiments, the WAP domain-containing polypeptide sequence ofthe fusion protein includes an Eppin polypeptide sequence or an aminoacid sequence that is derived from Eppin. These embodiments are referredto herein as “serpin_((a′))-Eppin fusion proteins. In some embodiments,the Eppin polypeptide sequence of the serpin-Eppin fusion proteinincludes a portion of the Eppin protein, such as for example, the WAPdomain or a sub-portion thereof. In a preferred embodiment, the Eppinpolypeptide sequence or an amino acid sequence that is derived fromEppin is or is derived from a human Eppin polypeptide sequence.

In some embodiments of the serpin-Eppin fusion proteins, the Eppinpolypeptide sequence or amino acid sequence derived from an Eppinpolypeptide is or is derived from one or more of the human Eppinpolypeptide sequences shown in GenBank Accession Nos. 095925.1,NP_(—)065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/orAAG00546.1.

In some embodiments, the serpin polypeptide of the serpin-WAP domainfusion protein includes at least the amino acid sequence of the reactivesite loop portion of the AAT protein. In some embodiments, the reactivesite loop portion of the AAT protein includes at least the amino acidsequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide ofthe serpin-WAP fusion protein includes at least the amino acid sequenceof a variant of the reactive site loop portion of the AAT protein. Insome embodiments, the variant of the reactive site loop portion of theAAT protein includes at least the amino acid sequence of SEQ ID NO:32 orSEQ ID NO:33. In some embodiments, the serpin polypeptide of theserpin-WAP domain fusion protein includes at least the full-length humanAAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. Insome embodiments the serpin polypeptide of the serpin-WAP domain fusionprotein includes human AAT polypeptide sequence that is at least 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or33.

In some embodiments, the serpin polypeptide of the serpin-WAP domainfusion protein includes the AAT polypeptide sequence is, or the aminoacid sequence derived from an AAT polypeptide is derived from, one ormore of the human AAT polypeptide sequences shown in GenBank AccessionNos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1,CAA25838.1, NP_(—)001002235.1, CAA34982.1, NP_(—)001002236.1,NP_(—)000286.3, NP_(—)001121179.1, NP_(—)001121178.1, NP_(—)001121177.1,NP_(—)001121176.16, NP_(—)001121175.1, NP_(—)001121174.1,NP_(—)001121172.1, and/or AAA51547.1.

In some embodiments, the serpin-WAP domain fusion protein can alsoinclude an Fc polypeptide or an amino acid sequence that is derived froman Fc polypeptide. These embodiments are referred to collectively hereinas “serpin-Fc-WAP domain fusion proteins.” In these embodiments, noparticular order is to be construed by this terminology. For example,the order of the fusion protein can be serpin-Fc-WAP domain, serpin-WAPdomain-Fc, or any variation combination thereof. The serpin-Fc-WAPdomain fusion proteins described herein include at least a serpinpolypeptide or an amino acid sequence that is derived from a serpin, WAPdomain-containing polypeptide or an amino acid sequence that is derivedfrom a WAP domain-containing polypeptide, and an Fc polypeptide or anamino acid sequence that is derived from an Fc polypeptide.

In some embodiments, where the serpin-WAP domain fusion protein includesan Fc polypeptide sequence, the Fc polypeptide sequence can have theamino acid sequence of SEQ ID NO: 3-7. In other embodiments, where theserpin-WAP domain fusion protein includes an Fc polypeptide sequence,the Fc polypeptide sequence can have at least 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NOs. 3-7. In some embodiments, theserpin-WAP domain fusion protein can also include an albuminpolypeptide, or an amino acid sequence that is derived from an albuminpolypeptide. These embodiments are referred to collectively herein as“serpin-albumin-WAP domain fusion proteins.” In these embodiments, noparticular order is to be construed by this terminology. For example,the order of the fusion protein can be serpin-albumin-WAP domain,serpin-WAP domain-albumin, or any variation combination thereof. Theserpin-albumin-WAP domain fusion proteins described herein include atleast a serpin polypeptide or an amino acid sequence that is derivedfrom a serpin, WAP domain-containing polypeptide, or an amino acidsequence that is derived from a WAP domain-containing polypeptide, andan albumin polypeptide, or an amino acid sequence that is derived froman albumin polypeptide.

In some embodiments where the serpin-WAP domain fusion protein includesan albumin polypeptide sequence, the albumin polypeptide sequenceincludes the amino acid sequence of SEQ ID NO: 14-15, described herein.In other embodiments, where the serpin-WAP domain fusion proteinincludes an albumin polypeptide sequence, the albumin polypeptidesequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the any one of the aminoacid sequences having SEQ ID NO: 14 or 15.

In some embodiments, the second polypeptide (Polypeptide 2) of theserpin fusion protein is an albumin polypeptide or is derived from analbumin polypeptide. These embodiments are referred to collectivelyherein as “serpin_((a′))-albumin fusion proteins.” The serpin-albuminfusion proteins described herein include at least a serpin polypeptideor an amino acid sequence that is derived from a serpin and an albuminpolypeptide or an amino acid sequence that is derived from an albuminpolypeptide. In addition this invention relates to serpin albuminbinding polypeptide fusion proteins, wherein the albumin is operablylinked to the serpin via an intermediate binding molecule. Herein, theserpin is non-covalently or covalently bound to human serum albumin.

In embodiments where the fusion protein of the invention includes analbumin polypeptide sequence, the albumin polypeptide sequence of thefusion protein is a human serum albumin (HSA) polypeptide or an aminoacid sequence derived from HSA. In some embodiments, the fusion proteinincludes a HSA polypeptide sequence having the following amino acidsequence:

(SEQ ID NO: 14)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

In embodiments where the fusion protein of the invention includes analbumin polypeptide sequence, the albumin polypeptide sequence of thefusion protein includes a human serum albumin polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 14.

In embodiments where the fusion protein of the invention includes analbumin polypeptide sequence, the albumin polypeptide sequence of thefusion protein fusion protein includes a domain 3 of human serum albuminpolypeptide sequence having the following amino acid sequence:

(SEQ ID NO: 15)EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA

In embodiments where the fusion protein of the invention includes analbumin polypeptide sequence, the albumin polypeptide sequence of thefusion protein includes a human serum albumin polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ IDNO: 15.

In some embodiments where the fusion protein of the invention includesan albumin polypeptide sequence, the fusion protein is linked to thehuman serum albumin via an intermediate albumin binding polypeptide. Thealbumin binding polypeptide can be an antibody or an antibody fragmentor derived from an antibody or antibody fragment. In a preferredembodiment, the albumin binding polypeptide or an amino acid sequencethat is derived from the antibody or antibody fragment is derived from achimeric, humanized, or fully human antibody sequence. The term antibodyfragment includes single chain, Fab fragment, a F(ab′)₂ fragment, ascFv, a scAb, a dAb, a single domain heavy chain antibody, and a singledomain light chain antibody. In addition, the albumin bindingpolypeptide can be an albumin binding peptide. Another embodiment of theinvention is a serpin albumin binding polypeptide fusion, wherein thealbumin binding polypeptide is domain 3 of Streptococcal protein G or asequence derived from domain 3 of Streptococcal protein G.

In some embodiments, the serpin polypeptide of the serpin_((a′))-albuminfusion proteins includes at least the amino acid sequence of thereactive site loop portion of the AAT protein. In some embodiments, thereactive site loop portion of the AAT protein includes at least theamino acid sequence of SEQ ID NO:1. In some embodiments, the serpinpolypeptide of the serpin-albumin fusion protein includes at least theamino acid sequence of a variant of the reactive site loop portion ofthe AAT protein. In some embodiments, the variant of the reactive siteloop portion of the AAT protein includes at least the amino acidsequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, theserpin polypeptide of the serpin-albumin fusion proteins includes atleast the full-length human AAT polypeptide sequence having amino acidsequence of SEQ ID NO: 2. In some embodiments the serpin polypeptide ofthe serpin-albumin fusion proteins includes human AAT polypeptidesequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the serpin-albumin fusionproteins includes the AAT polypeptide sequence or the amino acidsequence derived from an AAT polypeptide is or is derived from one ormore of the human AAT polypeptide sequences shown in GenBank AccessionNos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1,CAA25838.1, NP_(—)001002235.1, CAA34982.1, NP_(—)001002236.1,NP_(—)000286.3, NP_(—)001121179.1, NP_(—)001121178.1, NP_(—)001121177.1,NP_(—)001121176.16, NP_(—)001121175.1, NP_(—)001121174.1,NP_(—)001121172.1, and/or AAA51547.1.

In some embodiments, the fusion proteins are modified to increase orotherwise inhibit proteolytic cleavage, for example, by mutating one ormore proteolytic cleavage sites. In some embodiments, the fusionproteins are modified to alter or otherwise modulate an Fc effectorfunction of the fusion protein, while simultaneously retaining bindingand inhibitory function as compared to an unaltered fusion protein. Fceffector functions include, by way of non-limiting examples, Fc receptorbinding, prevention of proinflammatory mediator release upon binding tothe Fc receptor, phagocytosis, modified antibody-dependent cell-mediatedcytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC),modified glycosylation at Asn297 residue (EU index of Kabat numbering,Kabat et at 1991 Sequences of Proteins of Immunological Interest) of theFc polypeptide. In some embodiments, the fusion proteins are mutated orotherwise modified to influence Fc receptor binding. In someembodiments, the Fc polypeptide is modified to enhance FcRn binding.Examples of Fc polypeptide mutations that enhance binding to FcRn areMet252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (Kabat numbering,Dall'Acqua et at 2006, J. Biol Chem Vol 281(33) 23514-23524), orMet428Leu and Asn434Ser (M428L, N434S) (Zalevsky et at 2010 NatureBiotech, Vol. 28(2) 157-159). (EU index of Kabat et at 1991 Sequences ofProteins of Immunological Interest). In some embodiments the Fcpolypeptide portion is mutated or otherwise modified so as to disruptFc-mediated dimerization (Ying et at 2012 J. Biol Chem 287(23):19399-19408). In these embodiments, the fusion protein is monomeric innature.

The fusion proteins and variants thereof provided herein exhibitinhibitory activity, for example by inhibiting a serine protease such ashuman neutrophil elastase (NE), a chemotrypsin-fold serine protease thatis secreted by neutrophils during an inflammatory response. The fusionproteins provided herein completely or partially reduce or otherwisemodulate serine protease expression or activity upon binding to, orotherwise interacting with, a serine protease, e.g., a human serineprotease. The reduction or modulation of a biological function of aserine protease is complete or partial upon interaction between thefusion proteins and the human serine protease protein, polypeptideand/or peptide. The fusion proteins are considered to completely inhibitserine protease expression or activity when the level of serine proteaseexpression or activity in the presence of the fusion protein isdecreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% ascompared to the level of serine protease expression or activity in theabsence of interaction, e.g., binding, with a fusion protein describedherein. The fusion proteins are considered to partially inhibit serineprotease expression or activity when the level of serine proteaseexpression or activity in the presence of the fusion protein isdecreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%,75%, 80%, 85% or 90% as compared to the level of serine proteaseexpression or activity in the absence of interaction, e.g., binding,with a fusion protein described herein.

The fusion proteins described herein are useful in a variety oftherapeutic, diagnostic and prophylactic indications. For example, thefusion proteins are useful in treating a variety of diseases anddisorders in a subject. In some embodiments, the serpin fusion proteins,including, fusion proteins described herein, are useful in treating,alleviating a symptom of, ameliorating and/or delaying the progressionof a disease or disorder in a subject suffering from or identified asbeing at risk for a disease or disorder selected fromalpha-1-antitrypsin (AAT) deficiency, emphysema, chronic obstructivepulmonary disease (COPD), acute respiratory distress syndrome (ARDS),allergic asthma, cystic fibrosis, cancers of the lung,ischemia-reperfusion injury, including, e.g., ischemia/reperfusioninjury following cardiac transplantation, myocardial infarction,rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosingspondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes,bacterial infections, fungal infections, viral infections, pneumonia,sepsis, graft versus host disease (GVHD), wound healing, Systemic lupuserythematosis, and Multiple sclerosis.

Pharmaceutical compositions according to the invention include a fusionprotein of the invention, including modified fusion proteins and othervariants, along with a suitable carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of some embodiments of serpin-Fcfusion proteins according to the invention. The serpin can be located atany position within the fusion protein. Serpin-Fc fusion proteinincorporating more than one serpin polypeptide are also represented.

FIG. 1B is a photograph of a SDS-PAGE gel showing serum derived AAT(lane 1), AAT-Fc1 (lane 2, human IgG1 Fc), and AAT-EL-Fc1 (lane 3,Met351Glu, Met358Leu mutations within AAT, human IgG1 Fc).

FIG. 1C is a graph showing the inhibition of neutrophil elastaseactivity by AAT-Fc fusion proteins.

FIG. 1D is a photograph of a SDS-PAGE gel showing tetravalentAAT-Fc-AAT, having two AAT polypeptides per Fc polypeptide.

FIG. 1E is a graph showing the inhibition of neutrophil elastaseactivity by a tetravalent AAT-Fc-AAT fusion protein.

FIG. 1F is a graphing demonstrating the effect of low pH elution fromprotein A resin, wherein the NE inhibiting capacity of the AAT-Fc fusionprotein eluted at low pH is drastically reduced.

FIG. 1G is a graph showing that the double mutant, AAT-EL-Fc (Met351Glu,Met358Leu mutations) is resistant to H₂O₂ inactivation (conc.), comparedto wild type AAT and the single mutant AAT-EM-Fc (Met351Glu).

FIG. 1H is a graph depicting the serum clearance rates of serum derivedAAT (sdAAT) compared to AAT-Fc in rats dosed with 10 mg/kg protein (3rats/test protein). The half-life of AAT-Fc is substantially longer thanthat of sdAAT.

FIG. 2A is a schematic representation of some embodiments of theserpin-cytokine targeting fusion proteins of the invention. The serpincan be fused to either the heavy chain, the light chain, or both of anantibody. Serpin-cytokine receptor fusion proteins are also depicted.

FIG. 2B is a photograph of a SDS-PAGE gel showing the D2E7 antibody(lane 1), and the D2E7 antibody with-AAT fused to heavy chain (lane 2).

FIG. 2C is a graph showing the inhibition of neutrophil elastaseactivity by a D2E7 antibody fused to AAT. Serum derived AAT is shown asa positive control, whereas the D2E7 antibody alone is shown as anegative control for NE inhibition.

FIG. 3A is a schematic representation of some embodiments of theserpin-Fc-WAP fusion proteins.

FIG. 3B is a photograph of a SDS-PAGE gel showing AAT-Fc-ELAFIN (lane 1)and AAT-Fc-SLPI (lane 2).

FIG. 3C is a graph showing the inhibition of neutrophil elastaseactivity by an AAT-Fc-ELAFIN fusion protein and an AAT-Fc-SLPI fusionprotein. An AAT-Fc fusion protein and serum derived AAT are included forcomparison.

FIG. 4A is a schematic representation of some embodiments of the AAT-HSAfusion proteins.

FIG. 4B is a photograph of a SDS-PAGE gel showing an AAT-HSA fusion.

FIG. 4C is a graph showing the inhibition of neutrophil elastaseactivity by an AAT-HSA compared to serum derived AAT.

DETAILED DESCRIPTION OF THE INVENTION

Human neutrophil elastase (NE) is a chymotrypsin-fold serine protease,secreted by neutrophils during inflammation. Aberrant activity of NEresults in a progressive degradation of elastin tissues and the slowdestruction of the alveolar structures of the lungs leading to emphysemaand lung fibrosis (Lungarella et at 2008 Int. J. Biochem Cell Biol40:1287). Often, misguided NE activity is due to an imbalance of theprotease with its natural inhibitor, alpha1-antitrypsin (AAT). Thisimbalance can result from enhanced neutrophil infiltration into thelungs, as observed in the lungs of smokers and patients with CysticFibrosis (CF), or Acute Respiratory Distress Syndrome (ARDS).Conversely, a deficiency of AAT, usually as a result of a point mutationthat causes ATT to aggregate and accumulate in the liver, leaves thelungs exposed to unchecked NE activity. Individuals with AATdeficiencies are at increased the risk of emphysema, COPD, liverdisease, and numerous other conditions.

AAT deficiency affects approximately 100,000 Americans (according toestimates from the Alpha-1 Foundation), and many of the afflicted peopledie in their 30's and 40's. There are currently only a few FDA-approveddrugs for treatment of ATT deficiency (Prolastin®, Aralast™, Zemaira®,Glassia™). Each drug is the natural AAT derived from pooled humanplasma, which appears to be insufficient to meet the anticipatedclinical demand. Furthermore, these products have short serum half-lives(T_(1/2) of approximately 5 days) and require high dose (60 mg/kg bodyweight) weekly infusions. The current market for these drugs isestimated at approximately $400 million. The market for AAT-like drugsis likely substantially larger, based on the estimation that as many as95% of individuals with AAT-deficiencies go undiagnosed, and the factthat these drugs have the potential to be effective therapies forpathologies characterized by enhanced NE activity in individuals thatare not AAT-deficient (e.g., cystic fibrosis (CF), acute respiratorydistress syndrome (ARDS), smoking-induced emphysema and/or COPD).

AAT has been suggested to have broad spectrum anti-inflammatory activity(Tilg et at 1993 J Exp Med 178:1629-1636, Libert et al 1996 Immunol157:5126-5129, Pott et al, Journal of Leukocyte Biology 85 2009,Janciauskiene et at 2007 J. Biol Chem 282(12): 8573-8582, Nita et at2007 Int J Biochem Cell Biol 39:1165-1176). Recently, evidence hasmounted that AAT may be useful in treating numerous human pathologies,outside of the commonly suggested inflammatory pulmonary conditions.Human AAT has shown to protect mice from clinical and histopathologicalsigns of experimental autoimmune encephalomyelitis (EAE), suggesting itcould be a potential treatment of autoimmune diseases, such as multiplesclerosis or systemic lupus erythematosus (SLE) (Subramanian et al 2011Metab Brain Dis 26:107-113). Serum AAT has shown activity in rodentmodels of Graft Versus Host Disease (GVHD) (Tawara et at 2011 Proc.Natl. Acad. Sci. USA 109: 564-569, Marcondes et at 2011 Blood November3; 118(18):5031-9), which has led to a human clinical trial using AAT totreat individuals with Steroid Non-responsive Acute GVHD (NCT01523821).Additionally, AAT has been effective in animal models of type I and typeII diabetes, dampening inflammation, protecting islet cells fromapoptosis and enabling durable islet cell allograft (Zhang et at 2007Diabetes 56:1316-1323, Lewis et at 2005 Proc Natl Acad Sci USA102:12153-12158, Lewis et al 2008 Proc Natl Acad Sci USA105:16236-16241, Kalis et al 2010 Islets 2:185-189). Currently, thereare numerous early human clinical trials of type I diabetes using serumderived AAT products (NCT01183468, NCT01319331, NCT01304537).

The current serum-derived AAT products undergo extensive purificationand testing to ensure the removal of pathogenic viruses, however, therisk of transmission of infectious agents cannot be completelyeliminated. Moreover, serum is limited, which limits the productioncapacity of serum derived AAT. Attempts to address the concerns of serumderived products and production issues have been aimed at the expressionof recombinant AAT. However, after 20 years of work, the generation of atherapeutically viable recombinant AAT has yet to reach the market(Karnaukhova et al 2006 Amino Acids 30: 317). Like the plasma-derivedproducts, recombinant versions of AAT suffer from short serumhalf-lives, low production yields, and poor lung distribution.

The fusion proteins of the present invention have enhancedfunctionalities compared to the unmodified AAT molecule. The fusion ofan AAT polypeptide with a second polypeptide that interacts with theneonatal Fc receptor (FcRn), serves to increase the serum half-life,providing a much needed dosing benefit for patients. These FcRninteracting polypeptides of the fusion protein include immunoglobulin(Ig) Fc polypeptides derived from human IgG1, IgG2, IgG3, IgG4, or IgM,and derivatives of human albumin. In some embodiments, the fusionprotein incorporates mutations with the AAT portion that render themolecule more resistant to inactivation by oxidation. For exampleMet351Glu, Met358Leu (AAT-EL-Fc), demonstrates resistance inactivationby H₂O₂ oxidation (FIG. 1G). While AAT is a natural anti-inflammatoryprotein, some embodiments of the invention provide enhanced inflammationdampening capacity through the fusion of an AAT polypeptide and acytokine targeting polypeptide. The coupling of dual anti-inflammatoryfunctionalities from AAT and a second polypeptide, will provide more apotent therapeutic protein than either polypeptide on their own.Additionally, the coupling the anti-infective activity of AAT willmitigate the infection risk of most cytokine targeting biologics. Someembodiments provide for more potent anti-inflammatory and anti-infectiveproteins through the fusion an AAT-polypeptide and WAP domain containpolypeptide. The fusion proteins of the present invention are expectedto be a great therapeutic utility and be superior to the current serumderived AAT products.

To extend the half-life of recombinant AAT, recombinant DNA technologywas used to create a AAT gene fusion with the Fc domain of human IgG1,IgG2, IgG3, IgG4, IgM, or HSA, such that the expected protein productwould be AAT followed by an Fc domain ((AAT-Fc (IgG1), AAT-Fc (IgG2),AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc (IgM)) or AAT followed by HSA.While it was known that fusion of Fc domains of HSA to some proteins,protein domains or peptides could extend their half-lives (see e.g.,Jazayeri et al. BioDrugs 22, 11-26, Huang et al. (2009) Curr OpinBiotechnol 20, 692-699, Kontermann et al. (2009) BioDrugs 23, 93-109,Schmidt et al. (2009) Curr Opin Drug Discov Devel 12, 284-295), it wasunknown if an Fc domain or HSA fused to AAT would allow for properfolding and maintenance of NE inhibitory activity, or could extend thehalf-life of recombinant AAT. The fusion proteins of the presentinvention are shown to be potent inhibitors of NE, have extended serumhalf lives, and in some embodiments resistant to oxidation. In otherembodiments, the fusion proteins described herein have distinctproperties by the incorporation of other functional polypeptides,including cytokine targeting polypeptides, and WAP domain containingpolypeptides.

The fusion proteins described herein include at least a serpinpolypeptide or an amino acid sequence that is derived from a serpin anda second polypeptide. In some embodiments, for example, the inventionprovides a serpin polypeptide fused to human IgG1-Fc, IgG2-Fc, IgG3-Fc,IgG4-Fc, IgM-Fc, or HSA derivatives. The serpin-fusion described hereinare expected to be useful in treating a variety of indications,including, by way of non-limiting example, alpha-1-antitrypsin (AAT)deficiency, emphysema, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), allergic asthma, cysticfibrosis, cancers of the lung, ischemia-reperfusion injury, including,e.g., ischemia/reperfusion injury following cardiac transplantation,myocardial infarction, rheumatoid arthritis, septic arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type Iand/or type II diabetes, bacterial infections, fungal infections, viralinfections, pneumonia, sepsis, graft versus host disease (GVHD), woundhealing, Systemic lupus erythematosis, and Multiple sclerosis.

In some embodiments, the fusion proteins described herein include atleast an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequencethat is derived from AAT and second polypeptide. For example, theinvention provides alpha-1-antitrypsin (AAT) fused to human IgG1-Fc,IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA derivatives.

In some embodiments, the fusion proteins described herein include atleast a serpin polypeptide or an amino acid sequence that is derivedfrom a serpin polypeptide and a cytokine targeting polypeptide or anamino acid sequence that is derived from a cytokine targetingpolypeptide. For example, the invention provides serpin polypeptide or asequence derived from a serpin polypeptide fused to a human cytokinereceptor or derivative thereof. Another embodiment of the inventionprovides serpin polypeptide or a sequence derived from a serpinpolypeptide fused to a cytokine targeting antibody, e.g., ananti-cytokine antibody, or a sequence derived from of a cytokinetargeting antibody, e.g., an anti-cytokine antibody, or sequence derivedfrom a fragment of cytokine targeting antibody, e.g., a fragment of ananti-cytokine antibody. For example, the invention provides a serpinpolypeptide or a sequence derived from a serpin polypeptide fused to acytokine targeting polypeptide in which the cytokine targetingpolypeptide binds any of the following human cytokines TNFα, IgE, IL-12,IL-23, IL-6, IL-1α, IL-1β, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18,IL-27, or IL-32.

For example, in some embodiments, the cytokine targeting polypeptidetargets TNFα and includes any of the following TNFα-targetingpolypeptide or sequences derived from the following TNFα-targetingpolypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel® or ATN-103and ATN-192.

For example, in some embodiments, the cytokine targeting polypeptidetargets IgE and includes any of the following IgE-targeting polypeptideor sequences derived from the following IgE-targeting polypeptides:Xolair or FcεRI.

For example, in some embodiments, the cytokine targeting polypeptidetargets the shared p40 subunit of IL-12 and IL-23 and includes theStelara® polypeptide or sequences derived from the Stelara® polypeptide.

For example Stelara® the cytokine targeting polypeptide targets IL-13and includes the CDP7766 polypeptide or sequences derived from theCDP7766 polypeptide.

In some embodiments, the fusion proteins described herein include atleast a alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequencethat is derived from AAT and a cytokine targeting polypeptide or anamino acid sequence that is derived from a cytokine targetingpolypeptide. For example, the invention provides alpha-1-antitrypsininhibitor (AAT) fused a cytokine targeting polypeptide in which thecytokine targeting polypeptide binds any of the following humancytokines: TNFα, IgE, IL-6, IL-1α, IL-1β, IL-12, IL-17, IL-13, IL-23,IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

In some embodiments the cytokine targeting polypeptide binds a cytokinereceptor and prevents binding of the cytokine. For example, the presentinvention includes a serpin fused to a cytokine receptor targetingantibody. For example, the invention provides alpha-1-antitrypsininhibitor (AAT) fused a cytokine targeting polypeptide in which thecytokine targeting polypeptide binds the receptor of any of thefollowing human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1β, IL-12, IL-17,IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2,IL-18, IL-27, or IL-32.

For example, in some embodiments, the cytokine targeting polypeptidetargets the IL-6 receptor and includes the Actemra® polypeptide (asdescribed in patent publication EP0628639), or the ALX-0061 polypeptide(as described in WO2010/115998), or sequences derived from the Actemra®polypeptide, or ALX-0061 polypeptide.

For example, Actemra® the cytokine targeting polypeptide targets theIL-6 receptor and includes the tocilizumab polypeptide or sequencesderived from the tocilizumab polypeptide.

The targeting of inflammatory cytokines and immune-stimulating agents byprotein therapeutics has demonstrated clinical success in numerousinflammatory conditions. The most common proteins used as cytokinetargeting agents are the soluble cytokine receptors and monoclonalantibodies and fragments thereof. A significant drawback with targetingcytokines is the increased risk of infection in these patients, asevidenced by the TNFα targeting biologics, Remicade®, Humira®, Simponi®,Cimiza®, and Enbrel®, and the IL-12/23 p40 targeting antibody, Stelara®.This is likely to be a common problem of targeting inflammatorycytokines leading to immune suppression in patients. AAT and otherserpin proteins are interesting in that they demonstrate bothanti-infective and anti-inflammatory activities. Thus, theserpin-cytokine targeting polypeptide fusion proteins of this inventioncan dampen aberrant cytokine activities while alleviating the risk ofinfections.

In some embodiments, the fusion proteins described herein include aserpin polypeptide or an amino acid sequence that is derived from aserpin, a WAP domain-containing polypeptide or an amino acid sequencethat is derived from a WAP domain-containing polypeptide, and an Fcpolypeptide or an amino acid sequence that is derived from an Fcpolypeptide. For example, the invention provides a serpin polypeptide, aWAP domain-containing polypeptide and human IgG1-Fc, IgG2-Fc, IgG3-Fc,IgG4-Fc or IgM-Fc derivatives operably linked together in any functionalcombination. In some embodiments, the WAP domain containing protein ishuman SLPI or derived from human SLPI. In other embodiments, the WAPdomain containing protein is human ELAFIN or derived from human ELAFIN.In some embodiments, the fusion proteins described herein include atleast an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequencethat is derived from AAT and a SLPI polypeptide or an amino acidsequence that is derived from SLPI. In some embodiments, the fusionproteins described herein include at least an AAT polypeptide or anamino acid sequence that is derived from AAT and an ELAFIN polypeptideor an amino acid sequence that is derived from Elafin.

SPLI and Elafin are WAP domain containing proteins that display serineprotease inhibitory activity. Both of these proteins areanti-inflammatory in function. In addition these proteins possess broadanti-infective capacities toward numerous strains of bacteria, viruses,and fungi.

In some embodiments, the fusion proteins described herein include atleast a serpin polypeptide or an amino acid sequence that is derivedfrom a serpin and a human serum albumin (HSA) polypeptide or an aminoacid sequence that is derived from a HSA polypeptide. Furtherembodiments of invention include serpin-albumin binding polypeptidefusion proteins, wherein the albumin binding polypeptide is responsiblefor the association of the serpin and HSA. Thereby the inventionincludes both covalent and non-covalent linkages of the serpinpolypeptide and the HSA polypeptide or sequences derived from the serpinpolypeptide or a HSA polypeptide. For example, the invention provides aserpin polypeptide fused to human HSA, or HSA derivatives, or HSAbinding peptide or polypeptides.

In some embodiments, the fusion proteins described herein include atleast an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequencethat is derived from AAT and a HSA polypeptide or an amino acid sequencethat is derived from a HSA polypeptide. For example, the inventionprovides alpha-1-antitrypsin (AAT) fused to HSA or a fragment derivedfrom HSA, or an albumin binding polypeptide.

In some embodiments, the fusion proteins described herein include aserpin polypeptide or an amino acid sequence that is derived from aserpin, a HSA polypeptide or an amino acid sequence that is derived froma HSA polypeptide, and a WAP domain-containing polypeptide or an aminoacid sequence that is derived from a WAP domain-containing polypeptide.In some embodiments, the fusion proteins described herein include atleast an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequencethat is derived from AAT and a HSA polypeptide or an amino acid sequencethat is derived from a HSA polypeptide, and a SLPI polypeptide or aminoacid sequence derived from SLPI. In other embodiments, the fusionproteins described herein include at least an alpha-1-antitrypsin (AAT)polypeptide or an amino acid sequence that is derived from AAT and a HSApolypeptide or an amino acid sequence that is derived from a HSApolypeptide, and an Elafin polypeptide or amino acid sequence derivedfrom Elafin.

The fusion proteins of the present invention can be readily produced inmammalian cell expression systems. For example Chinese Hamster Ovary(CHO) cells, Human Embryonic Kidney (HEK) 293 cells, COS cells, PER.C6®,NS0 cells, SP2/0, YB2/0 can readily be used for the expression of theserpin fusion proteins described herein. Importantly, mammalian cellexpression systems produce proteins that are generally more optimal fortherapeutic use. In contrast to bacterial, insect, or yeast-basedexpression systems, mammalian cell expression systems yield proteinswith glycosylation patterns that are similar or the same as those foundin natural human proteins. Proper gylcosylation of a protein can greatlyinfluence serum stability, pharmacokinetics, biodistribution, proteinfolding, and functionality. Therefore, the ability to producetherapeutic proteins in mammalian expression systems has distinctadvantages over other systems. Furthermore, most of the mammalian cellexpression systems (e.g., CHO, NS0, PER.C6® cells) can be readily scaledin commercial manufacturing facilities to produce therapeutic proteinsto meet clinical demands. The fusion proteins described herein haveenhanced functionalities over the natural form of AAT and can beproduced in mammalian expression systems for clinical and commercialsupply. Some embodiments of the invention include a purification systemthat enables the isolation of serpin fusion proteins that retain theirability to inhibit NE. Importantly, the purification process of thepresent invention can be readily incorporated into today's commercialmammalian cell-based manufacturing processes.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients. The term patientincludes human and veterinary subjects.

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, buffers, excipients, and other agents that are incorporatedinto formulations to provide improved transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include a fusionprotein of the invention, are used to treat or alleviate a symptomassociated with a disease or disorder associated with aberrant serineprotease activity in a subject. The present invention also providesmethods of treating or alleviating a symptom associated with a diseaseor disorder associated with aberrant serine protease activity in asubject. A therapeutic regimen is carried out by identifying a subject,e.g., a human patient suffering from (or at risk of developing) adisease or disorder associated with aberrant serine protease activity,using standard methods, including any of a variety of clinical and/orlaboratory procedures. The term patient includes human and veterinarysubjects. The term subject includes humans and other mammals.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disease or disorderassociated with aberrant serine protease activity. Alleviation of one ormore symptoms of the disease or disorder associated with aberrant serineprotease activity indicates that the fusion protein confers a clinicalbenefit.

Methods for the screening of fusion proteins that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA), enzymatic assays, flow cytometry, and otherimmunologically mediated techniques known within the art.

The fusion proteins described herein may be used in methods known withinthe art relating to the localization and/or quantitation of a targetsuch as a serine protease, e.g., for use in measuring levels of thesetargets within appropriate physiological samples, for use in diagnosticmethods, for use in imaging the protein, and the like). The terms“physiological sample” and “biological sample,” used interchangeably,herein are intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. Included within the usage of the terms “physiologicalsample” and “biological sample”, therefore, is blood and a fraction orcomponent of blood including blood serum, blood plasma, or lymph.

In a given embodiment, fusion proteins specific for a given target, orderivative, fragment, analog or homolog thereof, that contain thetarget-binding domain, are utilized as pharmacologically activecompounds (referred to hereinafter as “Therapeutics”).

A fusion protein of the invention can be used to isolate a particulartarget using standard techniques, such as immunoaffinity, chromatographyor immunoprecipitation. Detection can be facilitated by coupling (i.e.,physically linking) the fusion protein to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

A therapeutically effective amount of a fusion protein of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between thefusion protein and its target that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the fusion proteinfor its specific target, and will also depend on the rate at which anadministered fusion protein is depleted from the free volume othersubject to which it is administered. Common ranges for therapeuticallyeffective dosing of an fusion protein or fragment thereof invention maybe, by way of nonlimiting example, from about 0.1 mg/kg body weight toabout 250 mg/kg body weight. Common dosing frequencies may range, forexample, from twice daily to once a month.

Where fusion protein fragments are used, the smallest inhibitoryfragment that specifically binds to the target is preferred. Forexample, peptide molecules can be designed that retain the ability tobind the target. Such peptides can be synthesized chemically and/orproduced by recombinant DNA technology. (See, e.g., Marasco et al.,Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation canalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. Alternatively, or inaddition, the composition can comprise an agent that enhances itsfunction, such as, for example, a cytotoxic agent, cytokine,chemotherapeutic agent, growth-inhibitory agent, an anti-inflammatoryagent or anti-infective agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fusion protein, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods.

Pharmaceutical Compositions

The fusion proteins of the invention (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the fusion roteinand a pharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 AAT-Fc Fusion Proteins and Variants

Exemplary, but non-limiting examples of AAT-Fc fusion proteins accordingto the invention include the following sequences. While these examplesinclude a hinge sequence and/or a linker sequence, fusion proteins ofthe invention can be made using any hinge sequence and/or a linkersequence suitable in length and/or flexibility. Alternatively fusionproteins can be made without using a hinge and/or a linker sequence. Forexample, the polypeptide components can be directly attached.

An exemplary AAT-Fc fusion protein is the AAT-hFc1 (human IgG1 Fc)described herein. As shown below, AAT polypeptide portion of the fusionprotein is underlined (SEQ ID NO: 2), the hinge region is shown innormal text (SEQ ID NO: 43), and the IgG-Fc polypeptide portion of thefusion protein is italicized (SEQ ID NO: 3).

AAT-hFc1 (human IgG1 Fc) (SEQ ID NO: 16)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKIHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An exemplary AAT-Fc fusion protein is the AAT-hFc2 (human IgG2 Fc),described herein. As shown below, AAT polypeptide portion of the fusionprotein is underlined (SEQ ID NO: 2), the hinge region is shown innormal text (SEQ ID NO: 44), and the IgG-Fc polypeptide portion of thefusion protein is italicized (SEQ ID NO: 4).

AAT-hFc2 (human IgG2 Fc) (SEQ ID NO: 17)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQk ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFc1 (human IgG1 Fc,Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptideportion of the fusion protein is underlined (SEQ ID NO: 34), the hingeregion is shown in normal text (SEQ ID NO: 43), the IgG-Fc polypeptideportion of the fusion protein is italicized (SEQ ID NO: 3), and theMet351Glu mutation is boxed, and the Met358Leu mutation is shaded ingrey.

AAT-MM-EL-hFcl(human IgG1 Fc, Met351G1u/Met358Leu) (SEQ ID NO: 18)

DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFc2 (human IgG2 Fc,Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptideportion of the fusion protein is underlined (SEQ ID NO: 34), the hingeregion is shown in normal text (SEQ ID NO: 44), the IgG-Fc polypeptideportion of the fusion protein is italicized (SEQ ID NO: 4), theMet351Glu mutation is boxed, and the Met358Leu mutation is shaded ingrey.

AAT-MM-EL-hFc2(human IgG2 Fc, Met351G1u/Met358Leu) (SEQ ID NO: 19)

QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

An exemplary AAT-Fc fusion protein is the AAT-MM-LL-hFc1 (human IgG1 Fc,Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptideportion of the fusion protein is underlined (SEQ ID NO: 35), the hingeregion is shown in normal text (SEQ ID NO: 43), the IgG-Fc polypeptideportion of the fusion protein is italicized (SEQ ID NO: 3), theMet351Leu mutation is shaded in black, and the Met358Leu mutation isshaded in grey.

AAT-MM-LL-hFcl(human IgG1 Fc, Met351Leu/Met358Leu) (SEQ ID NO:36)

DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

An exemplary AAT-Fc fusion protein is the AAT-MM:LL-hFc2 (human IgG2 Fc,Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptideportion of the fusion protein is underlined (SEQ ID NO: 35), the hingeregion is shown in normal text (SEQ ID NO: 44) the IgG-Fc polypeptideportion of the fusion protein is italicized (SEQ ID NO: 4), theMet351Leu mutation is shaded in black, and the Met358Leu mutation isshaded in grey.

AAT-MM: LL-hFc2(human IgG2 Fc, Met351Leu/Met358Leu) (SEQ ID NO:20)

QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

An exemplary AAT-Fc fusion protein is the AAT-hFc1-AAT (human IgG1),described herein. As shown below, AAT polypeptide portion of the fusionprotein is underlined (SEQ ID NO: 2), the hinge region is shown innormal text (SEQ ID NO: 43), the ASTGS linker is shown in normal text(SEQ ID NO: 45), and the IgG-Fc polypeptide portion of the fusionprotein is italicized (SEQ ID NO: 3).

AAT-hFc1-AAT (human IgG1) (SEQ ID NO: 21)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQ K

These exemplary AAT-Fc fusion proteins were made using the followingtechniques.

The gene encoding human AAT was PCR amplified from human liver cDNA(Zyagen). Specific point mutations within the gene encoding AAT or theFc region were generated by overlapping PCR. The AAT encoding gene wascloned in frame with a gene encoding the hinge region, followed by a CH2domain, and a CH3 domain of human IgG1, IgG2, IgG3, IgG4, or IgM into amammalian expression vector, containing a mammalian secretion signalsequence up stream of the AAT gene insertion site. These expressionvectors were transfected into mammalian cells (specifically HEK293 orCHO cells) and grown for several days in 8% CO₂ at 37° C. Therecombinant AAT-Fc fusion proteins were purified from the expressioncell supernatant by protein A chromatography. Importantly, a nearneutral pH buffer was used (Gentle Ag/Ab Elution Buffer, ThermoScientific) to elute the AAT-Fc fusion protein from the protein A resin.The AAT-Fc fusion protein can not be eluted from protein A resin using astandard low pH elution, as the ability of AAT to inhibit NE iscompromised following low pH treatment, likely due to a low pH mediatedoligomerization of AAT. FIG. 1F shows the effects of low pH elution onthe ability of AAT to inhibit neutrophil elastase. AAT-Fc fusion proteincan be purified either by protein A and a near neutral pH elutionbuffer, by CaptureSelect® Alpha-1 Antitrypsin affinity matrix (BAC BV).

The purified AAT-Fc fusion proteins were tested for activity bydetermining their ability to inhibit neutrophil elastase (NE). FIGS. 1Band 1D show a reducing SDS-PAGE gel of purified serum derived AAT(sdAAT) and AAT-Fc fusion proteins (FIG. 1B—lane 1: sdAAT, lane 2:AAT-Fc (SEQ ID NO: 16), lane 3: AAT-EL-Fc (SEQ ID NO: 18), FIG. 1DAAT-Fc-AAT (SEQ ID NO: 20). The proteins were visualized by stainingwith coomassie blue.

To monitor human Neutrophil Elastase (NE) activity a fluorescentmicroplate assay was used. Inhibitory activity was measured by aconcomitant decrease in the residual NE activity using the followingassay. This assay buffer is composed of 100 mM Tris pH 7.4, 500 mM NaCl,and 0.0005% Triton X-100. Human NE is used at a final concentration of 5nM (but can also be used from 1-20 nM). The fluorescent peptidesubstrate AAVP-AMC is used at a final concentration of 100 μM in theassay. The Gemini EM plate reader from Molecular Devices is used to readthe assay kinetics using excitation and emission wavelengths of 370 nmand 440 nm respectively, and a cutoff of 420 nm. The assay is read for10 min at room temperature scanning every 5 to 10 seconds. The Vmax persecond corresponds to the residual NE activity, which is plotted foreach concentration of inhibitor. The intercept with the x-axis indicatesthe concentration of inhibitor needed to fully inactivate the startingconcentration of NE in the assay. Human serum derived AAT (sdAAT) wasused as a positive control in these assays. The AAT-Fc fusion proteinsdisplay potent NE inhibitory activity as shown in FIG. 1C. The fusionwherein there are two AAT polypeptides fused to single Fc polypeptide(AAT-Fc-AAT) displays enhanced potency over both sdAAT and the AAT-Fcfusion protein comprising a single AAT polypeptide (FIG. 1E). Thesefindings presented here demonstrate for the first time the AAT can befused to an Fc region and maintain its ability to inhibit NE. Ofparticular interest, the AAT-Fc-AAT fusion protein was found to be amore potent NE inhibitor.

FIG. 1F demonstrates the resistance of the AAT-EL-Fc (M351E, M358L)fusion protein to inactivation by oxidation. AAT fusion proteins, AAT-Fc(wt), AAT-EL-Fc (M351E, M358L), and AAT-EM-Fc (M351E), were treated with33 mM H₂O₂ and compared to untreated fusion proteins in the NEinhibition assays. The inhibition of NE by AAT-EL-Fc was not comprisedby oxidation, converse to the other proteins tested.

Furthermore, AAT-Fc fusion protein displayed a longer serum half-life inrats compared to serum derived AAT (FIG. 1H). In these studies 3 ratsper each test protein were injected I.V. with 10 mg/kg of sdAAT orAAT-Fc. Serum sample were taken at various time points over a 48 period.The serum ATT concentration was using an ELISA. These findingsdemonstrate that the fusion proteins of the invention have improvedpharmacokinetic properties and are a superior therapeutic format overserum derived AAT, for treating numerous human inflammatory conditionsand infectious diseases.

Example 2 AAT-TNFα Targeting Molecule Fusion Proteins

The studies presented herein describe several, non-limiting examples ofrecombinant AAT derivatives comprising human AAT fused to an anti-TNFαantibody or a derivative of a TNFα receptor. These examples are providedbelow to further illustrate different features of the present invention.The examples also illustrate useful methodology for practicing theinvention. These examples do not and are not intended to limit theclaimed invention.

The fusion proteins below include cytokine targeting polypeptidesequences that are from or are derived from (i) the anti-TNFα antibodyD2E7 (also known as Adalimumab or Humira®), or (ii) the extracellulardomain of Type 2 TNFα Receptor (TNFR2-ECD). The AAT polypeptide portionof the fusion protein is underlined, the antibody constant regions(CH1-hinge-CH2-CH3, or CL) are italicized, and D2E7-VH, D2E7-VK, andTNFR2-ECD are denoted in bold text. While these examples include a hingesequence and/or a linker sequence, fusion proteins of the invention canbe made using any hinge sequence and/or a linker sequence suitable inlength and/or flexibility. Alternatively fusion proteins can be madewithout using a hinge and/or a linker sequence.

An exemplary AAT-TNFα fusion protein is D2E7-Light Chain-AAT (G₃S)₂Linker, described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted inbold text (SEQ ID NO: 37), the (G₃S)₂ linker is shown in normal text(SEQ ID NO: 46), and the antibody constant regions are italicized (SEQID NO: 38)

D2E7-Light Chain-AAT (G₃S)₂ Linker (SEQ ID NO: 22)DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

An exemplary AAT-TNFα fusion protein is D2E7-Light Chain-AAT ASTGSLinker, described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted inbold text (SEQ ID NO: 37), the ASTGS linker is shown in normal text (SEQID NO: 45), and the antibody constant regions is italicized (SEQ ID NO:38)

D2E7-Light Chain-AAT ASTGS Linker (SEQ ID NO: 23)DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

An exemplary AAT-TNFα fusion protein is D2E7-Heavy Chain-AAT (G₃S)₂Linker, described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted inbold text (SEQ ID NO: 39), the (G₃S)₂ linker is shown in normal text(SEQ ID NO: 46), and the antibody constant regions is italicized (SEQ IDNO: 40)

D2E7-Heavy Chain-AAT (G₃S)₂ Linker (SEQ ID NO: 24)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

An exemplary AAT-TNFα fusion protein is D2E7-Heavy Chain-AAT ASTGSLinker, described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted inbold text (SEQ ID NO: 39), the ASTGS linker is shown in normal text (SEQID NO: 45), and the antibody constant regions is italicized (SEQ ID NO:40)

D2E7-Heavy Chain-AAT ASTGS Linker (SEQ ID NO: 25)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

An exemplary AAT-TNFα fusion protein is TNFR2-ECD-Fc1-AAT(G₃S)₂ Linker,described herein. As shown below, the AAT polypeptide portion of thefusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is denoted inbold text (SEQ ID NO: 41), the hinge region is shown in normal text (SEQID NO: 43), the (G₃S)₂ linker is shown in normal text (SEQ ID NO: 46),and the antibody constant regions is italicized (SEQ ID NO: 42)

TNFR2-ECD-Fc1-AAT (G₃S)₂ Linker (SEQ ID NO: 26)LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

An exemplary AAT-TNFα fusion protein is TNFR2-ECD-Fc1-AAT ASTGS Linker,described herein. As shown below, the AAT polypeptide portion of thefusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is denoted inbold text (SEQ ID NO: 41), the hinge region is shown in normal text (SEQID NO: 43), the ASTGS linker is shown in normal text (SEQ ID NO: 45),and the antibody constant regions is italicized (SEQ ID NO: 42)

TNFR2-ECD-Fc1-AAT ASTGS Linker (SEQ ID NO: 27)LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

These exemplary AAT-TNFα targeting molecule fusion proteins were madeusing the following techniques.

The genes encoding the variable heavy (VH) and variable kappa (VK)regions of the anti-TNFα antibody, D2E7, were generated by genesynthesis. The D2E7-VH gene was cloned in frame with a gene encoding ahuman IgG1 antibody heavy chain constant region, consisting of a CH1domain, a hinge domain, a CH2 domain, and a CH3 domain, into a mammalianexpression vector, containing a mammalian secretion signal sequence upstream of the VH domain insertion site (D2E7-HC). The D2E7-VK gene wascloned in frame with a human antibody kappa light chain constant (CL)domain, into a mammalian expression vector, containing a mammaliansecretion signal sequence up stream of the VK domain insertion site(D2E7-LC). The AAT encoding gene and the adjacent 5′ linker sequencewere cloned in frame into the 3′ end of either, the CH3 domain of theD2E7 heavy chain gene (D2E7-HC-AAT), or the CL domain of the D2E7 lightchain gene (D2E7-LC-AAT) coding sequences in the above describedmammalian expression vectors. The extracellular domain of the TNFαReceptor 2 (TNFR2-ECD) was generated by gene synthesis and cloned inframe with a gene encoding the hinge region, followed by a CH2 domainand a CH3 domain of human IgG1 (hFc1) into a mammalian expression,containing a mammalian secretion signal sequence up stream of theTNFR2-ECD insertion site. The AAT encoding gene and the adjacent 5′linker sequence were cloned in frame into the 3′ end of the geneencoding TNFR2-ECD-hFc1 into a mammalian expression vector(TNFR2-ECD-hFc1-AAT).

The D2E7-HC-AAT expression vector was co-transfected with either theD2E7-LC or the D2E7-LC-AAT expression vector into mammalian cells(specifically HEK293 or CHO cells) to generate the D2E7 antibody withAAT fused to the C-terminus of the heavy chain or to the C-terminus ofboth the heavy chain and light chain, respectively. The D2E7-LC-AAT wasco-transfected with the D2E7-HC expression vector into mammalian cellsto generate the D2E7 antibody with AAT fused to the C-terminus of thelight chain. The TNFR2-hFc1-AAT expression vector was transfected intomammalian cells. Transfected cells were grown for several days in 8% CO₂at 37° C.

The recombinant AAT-TNFα targeting fusion proteins were purified fromthe expression cell supernatant by protein A chromatography. A nearneutral pH buffer was used (Gentle Ag/Ab Elution Buffer, ThermoScientific) to elute the AAT-TNFα targeting fusion proteins from theprotein A resin.

FIG. 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane 1) andvariant wherein AAT is fused to the heavy chain of D2E7 (lane 2). Theproteins were visualized by staining with coomassie blue.

The purified AAT-TNFα targeting molecule fusion proteins were tested foractivity by determining their ability to inhibit neutrophil elastase.Human serum derived AAT (sdAAT) was used as a positive control in theseassays. NE inhibitory assay were conducted as described above. FIG. 2Cdemonstrates relative to sdAAT, the AAT-TNFα targeting molecule fusionprotein shows similar inhibition of neutrophil elastase, indicating thatthe inhibitory capacity of AAT has not been compromised by its fusion toan antibody.

Example 3 AAT-Fc-SLPI and AAT-Fc-Elafin

The studies presented herein describe several, non-limiting examples ofrecombinant AAT derivatives comprising human AAT fused a WAP domaincontaining protein. These examples are provided below to furtherillustrate different features of the present invention. The examplesalso illustrate useful methodology for practicing the invention. The AATpolypeptide portion of the fusion protein is underlined, the Fc portionis italicized, and the WAP domain containing polypeptide is in boldfont. While these examples include a hinge sequence and/or a linkersequence, fusion proteins of the invention can be made using any hingesequence and/or a linker sequence suitable in length and/or flexibility.Alternatively fusion proteins can be made without using a hinge and/or alinker sequence. For example, the polypeptide components can be directlyattached.

An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc1-SLPI (human IgG1Fc), described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), the hinge region isshown in normal text (SEQ ID NO: 43), the ASTGS linker is shown innormal text (SEQ ID NO: 45), the Fc portion is italicized (SEQ ID NO:3), and the WAP domain containing polypeptide is in bold font (SEQ IDNO: 9)

AAT-hFc1-SLPI (human IgG1 Fc) (SEQ ID NO: 28)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKA

An exemplary AAT-Fc-Elafin fusion protein is AAT-hFc1-Elafin (human IgG1Fc), described herein. As shown below, the AAT polypeptide portion ofthe fusion protein is underlined (SEQ ID NO: 2), the hinge region isshown in normal text (SEQ ID NO: 43), the ASTGS linker is shown innormal text (SEQ ID NO: 45), the Fc portion is italicized (SEQ ID NO:3), and the WAP domain containing polypeptide is in bold font (SEQ IDNO: 12)

AAT-hFc1-Elafin (human IgG1 Fc) (SEQ ID NO: 29)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ

The genes encoding the SLPI and Elafin were PCR amplified from humanspleen cDNA (Zyagen). These genes were cloned into the mammalianexpression vectors of example 1, wherein the SLPI or Elafin gene wasinserted in frame with the AAT-Fc gene. These expression vectors weretransfected into mammalian cells (specifically HEK293 or CHO cells) andgrown for several days in 8% CO₂ at 37° C. The recombinant AAT-Fc-WAPdomain fusion proteins were purified from the expression cellsupernatant by protein A chromatography. A near neutral pH buffer wasused (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute theAAT-Fc-WAP domain fusion protein from the protein A resin.

FIG. 3B shows an SDS-PAGE gel of the AAT-Fc-WAP fusion proteins (lane 1AAT-Fc-Elafin, lane 2 AAT-Fc-SLPI). The proteins were visualized bystaining with coomassie blue. The purified AAT-Fc-WAP domain fusionproteins were tested for activity by determining their ability toinhibit neutrophil elastase. NE inhibitory assays were conducted asdescribed above. Human serum derived AAT (sdAAT) and the AAT-Fc fusionprotein were used as a positive control in these assays. Relative tosdAAT, the AAT-Fc-WAP targeting molecule fusion proteins displayenhanced potency of NE inhibition of neutrophil elastase (FIG. 3C).

Example 4 AAT-Albumin

The studies presented herein describe several, non-limiting examples ofrecombinant AAT derivatives comprising human AAT fused an albuminpolypeptide. These examples are provided below to further illustratedifferent features of the present invention. The examples alsoillustrate useful methodology for practicing the invention. Theseexamples do not and are not intended to limit the claimed invention. TheAAT portion is underlined and the albumin portion is italicized. Forexample, the polypeptide components can be directly attached.

An exemplary AAT-Albumin fusion protein is AAT-HSA, described herein. Asshown below, the AAT polypeptide portion of the fusion protein isunderlined (SEQ ID NO: 2), the ASTGS linker is shown in normal text (SEQID NO: 45), and the albumin polypeptide is italicized (SEQ ID NO: 14)

AAT-HSA (SEQ ID NO: 30)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ASTGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

An exemplary AAT-Albumin fusion protein is AAT-HSA Domain 3, describedherein. As shown below, the AAT polypeptide portion of the fusionprotein is underlined (SEQ ID NO: 2), ASTGS linker is shown in normaltext (SEQ ID NO: 45), and the albumin polypeptide is italicized (SEQ IDNO: 15)

AAT-HSA Domain 3 (SEQ ID NO: 31)EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA

The gene encoding human serum albumin (HSA) was PCR amplified from humanliver cDNA (Zyagen). A mammalian expression vector was generated,wherein gene encoding HSA or the domain 3 of HSA, was cloned in frame tothe 3′ end of the AAT encoding gene, containing a mammalian secretionsignal sequence up stream of AAT.

These expression vectors were transfected into mammalian cells(specifically HEK293 or CHO cells) and grown for several days in 8% CO₂at 37° C. The recombinant AAT-HSA fusion proteins were purified from theexpression cell supernatant using the CaptureSelect® Alpha-1 Antitrypsinaffinity matrix (BAC BV), wherein the binding buffer consisted of 20 mMTris, 150 mM NaCl, pH 7.4 and the elution buffer consisted of 20 mMTris, 2M MgCl₂ pH 7.4.

FIG. 4B shows an SDS-PAGE gel of the AAT-HSA fusion protein The proteinswere visualized by staining with coomassie blue. The purified AAT-HSAfusion proteins were tested for activity by determining their ability toinhibit neutrophil elastase. NE inhibitory assays were conducted asdescribed above. Human serum derived AAT (sdAAT) was used as a positivecontrol in these assays. Relative to sdAAT, the AAT-HS fusion proteindisplays similar potency of NE inhibition, demonstrating that the fusionto albumin does not dampen the capacity of AAT to inhibit NE (FIG. 4C.)

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated fusion protein comprising at leastone human serpin polypeptide operably linked to a second polypeptidewherein the second polypeptide comprises at least one the following: animmunoglobulin Fc polypeptide or an amino acid sequence that is derivedfrom an immunoglobulin Fc polypeptide; a cytokine targeting polypeptideor a sequence derived from a cytokine targeting polypeptide; a WAPdomain containing polypeptide or a sequence derived from a WAP domaincontaining polypeptide; or an albumin polypeptide or an amino acidsequence that is derived from a serum albumin polypeptide.
 2. Anisolated fusion protein comprising at least one alpha-1 antitrypsin(AAT) polypeptide operably linked to a second polypeptide of at leastone of the following: an immunoglobulin Fc polypeptide; a cytokinetargeting peptide; a human secretory leukocyte proteinase inhibitor(SLPI) polypeptide; and an immunoglobulin Fc polypeptide and a humanSLPI polypeptide, wherein the SLPI polypeptide comprises the WAP2 domainof human SLPI; or a human serum albumin (HSA) polypeptide.
 3. Anisolated fusion protein comprising at least one alpha-1 antitrypsin(AAT) polypeptide operably linked to a second polypeptide of at leastone of the following: an immunoglobulin Fc polypeptide; a cytokinetargeting peptide; a human Elafin polypeptide; a human Elafinpolypeptide and an immunoglobulin Fc polypeptide, wherein the Elafinpolypeptide comprises the WAP domain of human Elafin; or a human serumalbumin (HSA) polypeptide.
 4. The fusion protein of claim 1 wherein thehuman serpin polypeptide is a human alpha-1 antitrypsin (AAT)polypeptide or is derived from a human AAT polypeptide.
 5. The isolatedfusion protein of claim 4, wherein AAT polypeptide comprises the aminoacid sequence of SEQ ID NO:
 2. 6. The isolated fusion protein of claim4, wherein the AAT polypeptide comprises the reactive site loop of AATcomprising the amino acid sequence of SEQ ID NO: 1 or a mutated reactivesite loop of AAT comprising the amino acid sequence of SEQ ID NO: 32 or33.
 7. The isolated fusion protein of claim 4, wherein theimmunoglobulin Fc polypeptide is a human Fc polypeptide.
 8. The isolatedfusion protein of claim 7, wherein human Fc polypeptide is a human IgMpolypeptide or a human IgG Fc polypeptide.
 9. The isolated fusionprotein of claim 8, wherein the human IgG Fc polypeptide is a human IgG1polypeptide, a human IgG2 Fc polypeptide, human IgG3 Fc polypeptide, orhuman IgG4 Fc polypeptide.
 10. The isolated fusion protein of claim 1,wherein the immunoglobulin Fc polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 3, 4, 5, and7.
 11. The isolated fusion protein of claim 1, wherein the serpinpolypeptide and the immunoglobulin Fc polypeptide are operably linkedvia a hinge region, a linker region, or both a hinge region and linkerregion.
 12. The isolated fusion protein of claim 4, wherein the AATpolypeptide and the immunoglobulin Fc polypeptide are operably linkedvia a hinge region, a linker region, or both a hinge region and linkerregion.
 13. The isolated fusion protein of claim 11, wherein the hingeregion, the linker region or both the hinge region and the linker regioncomprise a peptide sequence.
 14. The isolated fusion protein of claim 1,wherein the fusion protein comprises an amino acid sequence selectedfrom SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, and SEQ ID NO:
 21. 15. The isolated fusion protein of claim 1,wherein the cytokine targeting polypeptide is a human cytokine receptorpolypeptide or a derivative of a human cytokine receptor polypeptide, acytokine targeting polypeptide that binds a cytokine receptor or acytokine targeting polypeptide binds a human cytokine.
 16. The isolatedfusion protein of claim 15, wherein the human cytokine receptorpolypeptide comprises multiple subunits.
 17. The isolated fusion proteinof claim 15, wherein the human cytokine receptor is a receptor for TNF,IgE, IL-12, IL-23, IL-6, IL-1, IL-1, IL-17, IL-13, the p40 subunit ofIL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
 18. Theisolated fusion protein of claim 17, wherein the cytokine receptor is areceptor for human TNF.
 19. The isolated fusion protein of claim 15,wherein the human cytokine is TNF, IgE, IL-12, IL-23, IL-6, IL-1, IL-1,IL-17, IL-13, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2,IL-18, IL-27, or IL-32.
 20. The isolated fusion protein of claim 19,wherein the cytokine is human TNF.
 21. The isolated fusion protein ofclaim 15, wherein the cytokine targeting polypeptide is an antibody oran antibody fragment.
 22. The isolated fusion protein of claim 21,wherein the antibody or antibody fragment is chimeric, humanized, orfully human.
 23. The isolated fusion protein of claim 1, where thefusion protein comprises an amino acid sequence selected from SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQID NO:
 27. 24. The isolated fusion protein of claim 1, wherein the WAPdomain containing polypeptide sequence comprises a SLPI polypeptidesequence comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, 9 and
 10. 25. The isolated fusion protein ofclaim 1, wherein the WAP domain containing polypeptide sequencecomprises an elafin polypeptide sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 11, 12 and 13.26. The isolated fusion protein of claim 1, wherein the fusion proteincomprises a serpin polypeptide, a WAP domain-containing polypeptide andan immunoglobulin Fc polypeptide such that at least two of the serpinpolypeptide, the WAP domain-containing polypeptide and theimmunoglobulin Fc polypeptide are operably linked via a hinge region, alinker region, or both a hinge region and linker region.
 27. Theisolated fusion protein of claim 2, wherein the fusion protein comprisesan AAT polypeptide, a SLPI polypeptide and an immunoglobulin Fcpolypeptide such that at least two of the AAT polypeptide, the SLPIpolypeptide and the immunoglobulin Fc polypeptide are operably linkedvia a hinge region, a linker region, or both a hinge region and linkerregion.
 28. The isolated fusion protein of claim 3, wherein the fusionprotein comprises an AAT polypeptide, an Elafin polypeptide and animmunoglobulin Fc polypeptide such that at least two of the AATpolypeptide, the Elafin polypeptide and the immunoglobulin Fcpolypeptide are operably linked via a hinge region, a linker region, orboth a hinge region and linker region.
 29. The isolated fusion proteinof claim 4, wherein the fusion protein comprises two AAT polypeptidesand an immunoglobulin Fc polypeptide.
 30. The isolated fusion protein ofclaim 29, wherein each of the two AAT polypeptides are operably linkedto the immunoglobulin Fc polypeptide via a hinge region, a linkerregion, or both a hinge region and linker region such that the fusionprotein has the structural arrangement from N-terminus to C-terminus asfollows: AAT polypeptide-immunoglobulin Fc polypeptide-AAT polypeptide.31. The isolated fusion protein of claim 26, wherein the hinge region,the linker region or both the hinge region and the linker regioncomprise a peptide sequence.
 32. The isolated fusion protein of claim 2,wherein the fusion protein comprises the amino acid sequence of SEQ IDNO:
 28. 33. The isolated fusion protein of claim 2, where the fusionprotein comprises the amino acid sequence of SEQ ID NO:
 29. 34. Thefusion protein of claim 1, wherein the albumin polypeptide is a humanserum albumin (HSA) polypeptide or is derived from a HSA polypeptide.35. The isolated fusion protein of claim 34, wherein HSA polypeptidecomprises the amino acid sequence of SEQ ID NO: 14 or the amino acidsequence of SEQ ID NO:
 15. 36. The isolated fusion protein of claim 34,wherein the HSA polypeptide comprises domain 3 of HSA.
 37. The isolatedfusion protein of claim 34, wherein the serpin is operably linked to HSAvia an albumin binding polypeptide.
 38. The isolated fusion protein ofclaim 2, wherein the AAT polypeptide is operably linked to HSA via analbumin binding polypeptide.
 39. The isolated fusion protein of claim37, wherein the albumin binding polypeptide is an antibody or antibodyfragment or derived from an antibody or antibody fragment.
 40. Theisolated fusion protein of claim 39, wherein the antibody or antibodyfragment or chimeric, humanized, or fully human.
 41. The isolated fusionprotein of claim 37, wherein the albumin binding polypeptide is apeptide.
 42. The isolated fusion protein of claim 47, wherein thealbumin binding polypeptide is Domain 3 of Streptococcal Protein G or asequence derived from Domain 3 of Streptococcal Protein G.
 43. Theisolated fusion protein of claim 1, wherein the serpin is covalentlylinked to a HSA polypeptide.
 44. The isolated fusion protein of claim 1,wherein the serpin is non-covalently linked to a HSA polypeptide. 45.The isolated fusion protein claim 1, where the fusion protein comprisesthe amino acid sequence of SEQ ID NO: 30 or
 31. 46. The isolated fusionprotein of claim 1, wherein the immunoglobulin Fc polypeptide ismodified to enhance FcRn binding.
 47. The isolated fusion protein ofclaim 1, wherein the immunoglobulin Fc polypeptide comprising at leastone of the following mutations: Met252Tyr, Ser254Thr, Thr256Glu,Met428Leu or Asn434Ser.
 48. A method of treating or alleviating asymptom of a disease or disorder associated with aberrant serineprotease expression or activity in a subject in need thereof, the methodcomprising administering a fusion protein according to claim
 1. 49. Amethod of treating or alleviating inflammation or a symptom of aninflammatory disease or disorder while reducing the risk of infection,in a subject in need thereof, the method comprising administering afusion protein according to claim
 1. 50. A method of reducing the riskof infection in a subject in need thereof, the method comprisingadministering a fusion protein according to claim
 1. 51. The method ofclaim 48, wherein the subject is a human.
 52. The method of claim 49,wherein the inflammatory disease or disorder is selected from thefollowing: emphysema, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), allergic asthma, cysticfibrosis, cancers of the lung, ischemia-reperfusion injury,ischemia/reperfusion injury following cardiac transplantation,myocardial infarction, rheumatoid arthritis, septic arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type Iand/or type II diabetes, pneumonia, sepsis, graft versus host disease(GVHD), wound healing, Systemic lupus erythematosus, and Multiplesclerosis.
 53. The method of claim 50, wherein the infection is selectedfrom bacterial infections, fungal infections, or viral infections.